This invention relates to a mass spectrometer for macromolecules.
In prior art mass spectrometry, various techniques have successfully been established to volatilize and ionize biological macromolecules: Fast Atom Bombardment (FAB) [1], Electro Spray Ionization (ESI) [2, 3] and Matrix Assisted Laser Desorption/Ionization (MALDI) [4, 5, 6, 7]. In the MALDI method, the macromolecules are embeded with low concentration in a matrix of material with high photon absorption. When illuminated with high intensity laser light, the matrix heats up rapidly and evaporates into a plasma. During evaporation momentum is transferred to the macromoleculs which are subsequently ionized in the plasma. Because the matrix plasma cools rapidly, most macromolecules remain intact. In prior art mass spectrometers the masses of those ionized macromolecules are determined with the time-of-flight (TOF) method [3, 7, 8], with the Fourier Transform Ion Cyclotron Resonance (FT-ICR) method [2, 5, 6] or with the single or multi quadrupol mass filter method [9, 10].
The disadvantage of prior art ionizing particle detectors used in mass spectrometers for macromolecules is the strong decrease of ionization efficiency for massive macromolecules owing to their decreasing particle velocities [11, 12]. In state of the art detectors for mass spectrometry, the accelerated macromolecule emits an electron on impact with the detector which is subsequently multiplied by electron multiplier techniques. The efficiency to emit said first electron depends on the velocity of the impacting particle [11] which for a massive macromolecule is small. This lack of detector efficiency can be compensated for in prior art mass spectrometers by increasing the flux of macromolecules, however by the expense of decreasing the overall system sensitivity. Generally, in prior art mass spectrometers the detection of macromolecules with masses larger than typically 50000 amu is inefficient. With the FT-ICR technique much larger masses can be detected, however, at the expense of large integration times of the order of one second, excluding applications where high throughput is required.
Mass spectrometry is used in biology for protein sequencing [9, 13] and protein identification [10] by measuring the mass distribution of protein-fragments. It is also considered to be a promising technique to increase the speed and to reduce the cost in DNA-sequencing [2, 3, 5, 6, 7, 8, 14]. The standard DNA-sequencing procedure is to separate an aliquot of DNA-fragments, prepared according to the Maxam-Gilbert and Sanger strategy, using the pulsed gel-electrophoresis technique [15, 16]. In this technique, the DNA-fragments are separated by their lengths according to their migration properties in a gel to which an electrical field is applied. The spatially separated bands of DNA-fragments are conventionally recorded by auto-radiography and fluorescent techniques.
The disadvantage of said gel-electrophoresis technique is the slow sequencing rate and the poor mass resolution for very large DNA-fragments. The disadvantage of prior art mass spectrometers for high rate DNA-sequencing is the low sensitivity of ionizing detectors for DNA-fragments consisting of more than 100 bases, making inaccessible the increase in DNA-sequencing rate which is possible with mass spectrometry.